Physiology and regulation depend critically on proteins binding functional partners and avoiding nonfunctional associations. For each complex, the relative affinities are tuned to the concentrations of interacting pairs, the relative affinities of competing associations and the lifetimes required for function. This balance between binding affinity and specificity is a major thread in biology. To test hypotheses of the origins of binding specificity, we will explore the basis for recognition of a widespread motif, the alpha-helix. Using new methods, we will test the hypothesis that the protein structural flexibility and polymorphism play crucial roles in helix recognition. This research has four specific aims: 1. Determine crystal structures of TRAP coiled coils and the CaM:iNOS complex. 2. Use a new crystallographic method called Ringer to define the distributions of alternate rotamers in CaM:peptide complexes, coiled coils and other proteins. 3. Implement a direct electron-density sampling method, tau value analysis, to define the distributions of structural polymorphism in CaM:peptide complexes. 4. Test the hypothesis that polymorphism and flexibility correlate with binding thermodynamics in three CaM:peptide complexes by comparing the structural polymorphism in X-ray crystal structures, NMR measurements of flexibility and measurements of binding energetics. Building strongly on our previous work, we will extend the insights gained in coiled-coil systems to study helix recognition by CaM, the principal Ca2+ sensor in eukaryotes. Our preliminary results, including two new methods to analyze alternate structures in crystallographic electron density maps, establish the feasibility of all four aims. These studies afford a unique opportunity to correlate crystallographic and NMR measurements of polymorphism and dynamics. We will address an important outstanding mystery about CaM: How does the protein recognize ~200 different sequences and still exclude nonfunctional targets? By providing tools to develop a new view of structural ensembles using X-ray crystallography, our research will have a broad impact in structural biology, drug discovery and biomedical research.